The Expression Level of BRCA2 and Its Changes during Chemotherapy in Patients with Different Pathological Types of Mammary Cancer.

Background
We aimed to investigate the expression level of breast cancer susceptibility gene 2 (BRCA2) and its changes during chemotherapy in patients with different pathological types of mammary cancer (MC).


Methods
Overall, 102 patients treated in Affiliated Tumor Hospital of Guangxi Medical University, China from April 2013 to August 2017 were enrolled as experimental group, including 58 patients with noninvasive MC (group A) and 44 with invasive MC (group B). Fifty healthy volunteers at the same time were enrolled as control group. The relative expression of BRCA2 in the blood of MC patients was detected by real-time fluorescence quantitative PCR (FQ-PCR).


Results
In the experimental group, the expression level of BRCA2 in group A was higher than that in group B before chemotherapy (P<0.001); the expression level in group A and group B 1 month after chemotherapy was higher than that before chemotherapy (P<0.001); the expression level in the both groups 3 months after chemotherapy was higher than that 1 month after chemotherapy (P<0.001); the expression level of BRCA2 in blood of group A increased gradually before, 1 month and 3 months after chemotherapy (P<0.001). The expression level of BRCA2 in blood of group B increased gradually at the same time points (P<0.001).


Conclusion
BRCA2 is over-expressed in noninvasive MC patient and under-expressed in invasive MC patient. And it can be used as an index for monitoring the condition of MC patients with different pathological types during chemotherapy.

countries and large cities (2). MC has the characteristics of high incidence, high number of new cases, high mortality rate and low diagnostic rate, which seriously endangers the health of women and consumes a large amount of health resources every year (3). In recent years, modern medical technology in China has been continuously developed and improved in the treatment of MC, but the disease monitoring and 5-year survival rate of patients with MC have not been substantially improved (4). Therefore, it is of great clinical significance and value to explore the occurrence and development mechanism of MC, and to find out an effective MC disease monitor. Breast cancer susceptibility gene 2 (BRCA2) is mapped on 13q12, and has 27 exons and encodes a protein containing 3418 amino-acid residues, and has 8 internal repeats known as BRC sequences. BRCA2 protein is located in the nucleus and often related to the repair of DNA (5). BRCA2 expression is not only associated with the incidence of MC and ovarian cancer, but also affects the incidence of pancreatic cancer and prostate cancer (6). At present, it is believed that the variation of BRCA2 may lead to the decline in the stability of BRCA2 protein structure, promote the abnormal proliferation of cells, prevent the normal differentiation of the cells and eventually lead to the occurrence of the tumor, which is one of the mechanisms that lead to the occurrence of MC (7). This gene is a tumor suppressor gene that encodes tumor suppressor protein and inhibits tumor growth by regulating tumor production (8). Therefore, the expression of BRCA2 in serum of patients with different pathological types of MC and the changes of BRCA2 expression in blood before and after chemotherapy were discussed in this study to explore the monitoring value of BRCA2 in patients with different pathological types of MC during chemotherapy, and to provide reference for clinical practice.

Clinical data
Overall, 102 pathologically confirmed MC patients treated in Affiliated Tumor Hospital of Guangxi Medical University, China from April 2013 to August 2017 were randomly selected as experimental group, with an average age of (57.4±11.6) years. According to different pathological types, the experimental group was divided into two subgroups: noninvasive MC patients (group A, n=58) and invasive MC patients (group B, n=44). At the same time, 50 healthy volunteers with an average age of (55.1±14.2) years undergoing physical examination in our hospital were collected as control group. This study was approved by the Ethics Committee of the Institute, and all subjects were informed and agreed to participate in the clinical study and signed fully informed consent.

Inclusion and exclusion criteria
Inclusion criteria: Patients diagnosed with MC by clinicopathological diagnosis in our hospital; Age≥18 yr; No congenital hereditary disease; No radiotherapy, chemotherapy and other anticancer treatment before sampling. Exclusion criteria: Patients who had taken antibiotics within 3 months before sampling; patients with liver insufficiency; patients with autoimmune system deficiency; patients with other tumors; patients with cardiovascular disease; patients with recurrent MC.

Baseline data in experimental group and control group
There was no significant difference in age, fertility, amenorrhea, menarche time, smoking, body mass index (BMI) and other general clinical baseline data between the two groups. There were significant differences in tumor diameter, lymph node metastasis, skin viscosity and tumor differentiation degree and other pathological features between the subgroups (group A and group B) of the experimental group (P<0.05) ( Table 2).

Detection of BRCA2 expression in serum of experimental group and control group
BRCA2 was under-expressed in the blood of the experimental group (2.83±1.42) and was overexpressed in the blood of the control group (8.27±1.82). There was statistical difference between the two groups (t=20.17 P<0.001) (Fig. 1).

Detection of BRCA2 expression in experimental group with different clinical features
The expression of BRCA2 was not statistically different from age, fertility, amenorrhea, menarche time, smoking, BMI between the two groups.
There were significant differences in the expression of BRCA2 with tumor diameter, lymph node metastasis, skin viscosity and tumor differentiation degree (P<0.001) ( Table 3).

Detection of expression of BRCA2 in experimental group with different pathological types before chemotherapy
According to different pathological types, the experimental group was divided into two subgroups: noninvasive MC patients (group A, n=58) and invasive MC patients (group B, n=44).
The BRCA2 was under-expressed in group B before chemotherapy (1.78±0.37) and the expression of BRCA2 in group A (3.23±1.02) was higher than that in group B, there was statistical difference between the two groups (t=8.983, P<0.001) (Fig. 2).

Fig. 2:
The expression of BRCA2 in subgroups (Group A and group B) of the experimental group detected by FQ-PCR showed that the expression in the blood of group A was higher than that of group B (P<0.001). Note: * represented that the difference between the group A and group B was significant (t=8.983, P<0.001)

Expression of BRCA2 in subgroups (Group A and group B) of the experimental group after chemotherapy
The expression of BRCA2 in the group A before, 1 month and 3 months of chemotherapy showed an increasing trend, and the difference was statistically significant (P<0.001); the expression of BRCA2 in group B at the same time points showed an increasing trend, and the difference was statistically significant (P<0.001) ( Table 4).

Multivariate logistic regression analysis in the experimental group
Further multivariate analysis was performed on variables that were meaningful by univariate analysis, and the results showed that tumor diameter, lymph node metastasis, skin viscosity and tumor differentiation degree were selected as regression models (P<0.05). Tumor diameter, lymph node metastasis and skin viscosity were the risk factors affecting the expression level of BRCA2, and tumor differentiation was the protective factor (Table 5 and Table 6).

Discussion
With the development of economy and the deterioration of environment, the incidence of various kinds of malignant tumors is increasing. MC, one of the most common malignant tumors in women especially in urban women, is the major diseases threatening the health of women and has a high incidence and morbidity rate (9). Its incidence is second only to lung cancer (10), and is rising year by year, becoming the fastest (11) in all malignant tumors. The MC patients have a poor prognosis and a low 5-year survival rate (12), and there is no good disease monitoring indicator for evaluation of the status of chemotherapy. The occurrence, development and metastasis of MC has a great correlation with oncogene and tumor suppressor genes (13). Therefore, exploring the mechanism of the occurrence and development of MC and finding effective indicators of MC disease monitoring indicator is the focus of clinical research at the present stage. At present, epidermal growth factor receptor (EGFR) (14) is a common MC disease monitoring indicator, which has a unstable expression.
Some studies have shown that the expression of EGFR in MC may also be related to the decrease of degradation after activation, and the deletion of some domains of EGFR can lead to the destruction of receptor downregulation mechanism, activation of abnormal signal transduction pathway, inhibition of apoptosis and so on (15). Tumor marker 153 (CA153) is also known as one of the disease monitoring indicators (16), however, it lacks sufficient specificity and sensitivity, and has no obvious changes in serum of early MC patients, which is often similar to other benign diseases (17). Therefore, finding a stable, effective and sensitive MC disease monitor is a hot spot in the field of medicine. BRCA2, the second MC susceptibility gene (18) found in recent years, is expressed in many tissues, and the expression is the highest in mammary gland and thymus and slightly lower in lung, ovary and spleen (19). Normal BRCA2 protein is located in the nucleus and involved in the repair of DNA. The expression pattern of BRCA2 gene is similar to that of breast cancer susceptibility gene 1 during cell amplification, that is, the transcription of BRCA2 gene cannot be detected in resting cells (20). The expression of BRCA2 in the rapidly proliferating cells increase significantly and has cell cycle dependent (21). These results suggest that BRCA2 plays an important role in the regulation of cell growth. By comparing the expression of BRCA2 between the experimental group and the control group, it was found that BRCA2 was under-expressed in the serum of the experimental group and overexpressed in the serum of the control group. The experimental group was divided into two groups (group A and group B) according to pathological classification. The comparison between the two groups found that the expression of BRCA2 in the serum of noninvasive MC in group A was higher than that of invasive MC in group B. The expression of BRCA2 in serum of group A and group B 1 month after chemotherapy was higher than that before chemotherapy. The expression of BRCA2 in serum of the both groups 3 months after chemotherapy was higher than that 1 month after chemotherapy. Further multivariate analysis showed that tumor diameter, lymph node metastasis and skin viscosity were the risk factors affecting the expression level of BRCA2, and tumor differentiation was the protective factor. The results showed that the high expression level of BRCA2 was associated with high tumor differentiation and small tumor diameter, and the low expression level of BRCA2 with low tumor differentiation and large tumor diameter. The results of Kong et al (22) suggested that the increased mutation rate of BRCA2 increased the susceptibility to breast, ovarian and prostate cancer, which indicates that that BRCA2 is involved in the development of cancer cells in patients with MC, and has some value in the monitoring of MC. The subjects in this study were screened strictly according to the inclusion and exclusion criteria, and the chemotherapeutic drugs of MC were the products of Bristol Myers Squibb, which ensured the reliability of the results. However, there are still some defects in this study. For example, there were fewer patients included in the study; more MC patients with different pathological types should be collected; the relationship between BRCA2 and other clinical symptoms in MC patients was not further studied in this study. However, we will improve the study by following up the patients regularly according to the data of the patients in the experimental group and analyzing the results after the test.

Conclusion
Compared with normal volunteers, BRCA2 is under-expressed in the blood of noninvasive MC patients and invasive MC patient. The expression level of BRCA2 increases with the increase of chemotherapy time in MC patients. BRCA2 can be used as a monitoring indicator for patients with different pathological types of MC during chemotherapy, which is worthy of widespread promotion in clinical practice.

Ethical considerations
Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.